Efficacy of Commercial Bovine Serum as a Component of Media Used to Grow Cooperia punctata In vitro

Abstract

Several commercial serums-agamma calf, bovine, fetal bovine, from calves 10 days to 6 months old, and from calves younger than 10 days-were incorporated into Ae medium (Leland, 1963, 1965). Each serum's efficacy in promoting in vitro growth and development of Cooperia punctata, a nematode parasite of ruminants, was compared with medium Ae containing from a donor calf. Culture tubes inoculated with exsheathed, 3rd-stage C. punctata larvae were maintained in a roller drum at 38.5 C for 35 or 56 days; some cultures were transferred weekly into new media and some were not. At termination, worms were preserved, measured, and counted, and data for each medium were compared and evaluated. All serums tested, when used as a component of Ae, promoted growth and development of C. punctata to the adult stage. Bovine was least effective as a substitute for obtained from a donor calf. Although medium containing fetal bovine was unsatisfactory because it tended to precipitate after thawing, it resulted in good growth and development during the first 3 weeks of culture. A minus gamma globulin (agamma serum) is our choice as a substitute for serum; this resulted in a clear medium and L, from eggs laid in vitro were observed after 28 days. Living worms were observed in all media in which worms were grown for 56 days without transfer, but weekly transfers into new media produced better cultures in tubes maintained that long. After 35 days in culture, a negative correlation between size of inoculum and percentage of worms reaching the adult stage was noted, although no correlation was observed between size of inoculum and size of adults. Largest adults were produced in cultures maintained for 56 days but transferred weekly into new media. Largest females were produced in media containing either from a donor calf (Ae) or commercial fetal (AeF); largest males were produced in AeF. The parasitic stages of Cooperia punctata grow well in medium Ae which normally contains from calves; omitting the results in fewer and smaller adult worms, and females so grown produce no eggs (Leland, 1963). In contrast, nonparasitic stages of C. punctata (L1 and L2) do not develop in medium containing (Leland, 1967b). If a readily available commercial could be substituted for helminth-free serum it would facilitate experimentation using the in vitro culture system, and also might simplify comparisons of studies from different laboratories. Thus we tested the efficacy of Ae medium in which various commercially available bovine serums were substituted for obtained from